The present invention relates generally to matrices for conducting messenger RNA (mRNA)-affinity chromatography. More specifically, the present invention relates to a paper matrix which may be used for both mRNA-affinity chromatography and as a priming matrix for generating matrix-bound complementary DNA (cDNA) from the mRNA bound to the matrix.
An organism's traits are encoded in DNA. The information for a specific protein, associated with a particular trait or characteristic, is converted into a second polynucleotide, mRNA, by a process called transcription. This specific bit of information is then translated into the particular protein. Different cells in an organism are specialized to produce only a portion of the proteins encoded in the total DNA complement. Some cells produce as much as 1-2 percent of their total protein as a single species. Generally, mRNA levels reflect this same bias. Hence, if one can start with a population of cells that are preferentially producing the protein of interest and one can produce a double-stranded DNA copy from the mRNA, the gene cloning will have the advantage of starting with a DNA population highly enriched in the desired gene. Moreover, the mRNA that accumulates in the cell and that is subsequently translated into protein is a mature form lacking introns. The double stranded DNA that results also lacks introns and, therefore, contains the amino acid information in an uninterrupted form.
mRNA affinity-chromatography is based on the tendency of complementary, single-stranded nucleic acids to form a double-stranded or duplex structure. Fortunately for the molecular biologist, an enzyme associated with certain animal viruses is capable of using mRNA as a template to generate a complementary piece of DNA. Eukaryotic mRNA typically has a run of adenylic acid residues at its 3' terminus so that a short oligomer of deoxythymidylate can be used as a primer to initiate enzymatic synthesis of cDNA. Because this enzymatic process is based on RNA templates, which is opposite from normal cellular transcription, these enzymes have been named reverse transcriptases. Cloning based on this initial conversion of mRNA to cDNA is termed cDNA cloning. The single-stranded cDNA can be converted into a double stranded DNA using a combination of ribonuclease H and DNA polymerase I. The former degrades the majority of mRNA in the RNA-DNA hybrid leaving short RNA fragments which can act as primers for DNA polymerase I to synthesis of the second DNA strand. This process results in a double-stranded cDNA. The newly-synthesized double-stranded DNA can be cloned into an appropriate plasmid vector for the gene sequence, and introduced and expanded in a typical bacterial host cell.
While there are many cDNA cloning schemes, all start with mRNA enriched for the particular sequence of interest and use enzymatic tools for converting mRNA into double-stranded cDNA. The resulting cDNA clones are typically low yield and must be amplified before identification and verification. The most widely employed method for amplifying cDNA is the polymerase chain reaction (PCR) described in U.S. Pat. Nos. 4,683,202 and 4,683,195, which are hereby incorporated by reference. Amplification of cDNA is accomplished through repeated cycles of DNA synthesis using nucleic acid polymerase enzymes. The steps typically include using Klenow, a large subunit of DNA polymerase I, or any number of heat stable DNA polymerases, i.e., Taq I, in combination with gene specific primers and a DNA template of chromosomal or cDNA origin annealing the primers to the DNA template, in which the enzyme extends the primer sequence by DNA polymerization; and heating the resultant duplexed DNA product to denature and separate the strands, thereby making the strands available for a next cycle of primer annealing and extension. This cycle is repeated continuously until the DNA sequence is present in sufficiently high concentrations to permit identification.
Identification of a cDNA clone typically depends upon hybridization of the clone with labeled oligonucleotides specific for a desired gene or screening of the cells transformed by the hybrids with antibodies specific for the desired protein.
The first step in nucleic acid replication and identification is to sequester mRNA. Gilham first demonstrated the feasibility of isolating polynucleotides by affinity chromatography using oligonucleotide ligands of complementary sequence. A cellulose fiber column matrix was used with a covalently bound homopolymer oligonucleotide ligand. Gilham, P. T., J. Amer. Chem. Soc., 86:4982, 1964; Gilham, P. T., Biochemistry, 7:2809, 1968. In Methods in Enzymology, 21(PtD):191-197, 1971entitled "Resolution of Nucleic Acids," P. T. Gilham published an article entitled "The Covalent Binding of Nucleotides, Polynucleotides, and Nucleic Acids to Cellulose" disclosing the binding of polynucleotides to cellulose through phosphodiester linkages at the 5' or 3' terminals of the polynucleotides. Gilham illustrates the synthesis of thymidine, deoxycytidine and deoxyadenosine polynucleotide celluloses.
Aviv, H. and Leder, P., Proc. Natl. Acad. Sci. USA, 69:1408, 1972 used a cellulose fiber column matrix for purifying eukaryotic mRNA using oligo-deoxythymidine (oligo-d(T) cellulose. Similar purification schemes have been developed using either an agarose or sepharose dextran polymer matrix with the RNA ligand, polyuridylic acid (poly-U). Conventional mRNA purification methods are performed by column affinity-chromatography using one of these matrices. These conventional methods typically involve forming a duplex between the 3' terminus polyadenylated regions of the mRNA or viral RNA and the oligonucleotide ligand of the oligo-dT or poly-U. High concentrations of monovalent cation drive duplex formation and stability at room temperature. Since the oligonucleotide ligand is bound to an insoluble matrix, the annealed mRNA remains bound to the matrix as long as the column buffer is maintained at a high ionic strength. The mRNA is eluted from the column matrix by denaturing the mRNA-ligand binding duplex by decreasing the ionic strength of the column buffer and raising the column temperature. Thus, mRNA affinity chromatography consists generally of the steps of annealing mRNA to the ligand using high ionic strength buffers, e.g., 0.5 M NaCl, washing the non-polyadenylated RNA from the column matrix with the same high ionic strength buffer, denaturing the mRNA-ligand binding complex and eluting the mRNA by washing the column matrix with a low ionic strength buffer, e.g., 10 mM Tris/1 mM EDTA (TE buffer).
The use of columns for mRNA affinity chromatography carries with it some important disadvantages. Column chromatography requires specialized equipment, column preparation and mRNA purification is often tedious, time consuming and complex. Packed columns often do not permit large RNA to move freely through the matrix resulting in exclusion and trapping of non-ligand bound RNA within the column matrix. Finally, packed columns often exclude or trap the RNA molecules which hinders the adaptation of this type of chromatography to an automatic format.
Many of these problems have been addressed by attempts to alter the geometry of the packed column by flattening the three dimensional nature of the matrix. The flatter matrix decreases the distance the RNA must diffuse either to interact with the ligand or be freed from the matrix. Wreschner, D. H. and Herzberg, M., Nucleic Acids Res., 12:1349-1359, 1984 disclosed a poly U ligand substituted paper matrix in which the RNA ligand was bound via diazonium linkage with a paper bound arylamine. This poly U paper had a very high affinity for poly A containing nucleic acids. Werner, D., Chemla, Y. and Herzberg, M., Anal. Biochem., 141:329-336, 1984 employed the messenger affinity paper of Wreschner and Herzberg to develop a more simplified process for separating poly-A RNA from total cytoplasmic RNA. Amersham Corporation (Arlington Heights, Ill.) manufactures a paper matrix under the trademark HYBOND-MAP; the only commercially available flat matrix for mRNA affinity chromatography. HYBOND-MAP is a paper matrix composed of a poly-U ligand bound to arylamine substituted cellulose paper. Purifying mRNA using HYBOND-MAP is a simple procedure which does not require specialized equipment. The annealing capacity for mRNA of a 1 cm.sup.2 piece of HYBOND-MAP is about 20 .mu.g which is sufficient for most routine analyses. Several samples can be processed simultaneously using separate pieces of poly-U paper. The poly-U ligand used by Wreschner and Herzberg, Werner, Chemla and Herzberg and in the Amersham HYBOND messenger affinity paper is, however, degradable by RNAse or by conditions of high pH, and is, therefore, unsuitable for processes which require alkaline denaturation. The HYBOND messenger RNA affinity paper cannot be easily regenerated for reuse. While the poly-U ligand can be used to prime cDNA synthesis, the utility of the resulting solid phase cDNA is severely limited because of the underlying RNA linkage to the paper matrix. This limitation renders the poly-U papers unsuitable for use as a priming matrix for mRNA-cDNA synthesis for subsequent use as a solid-phase template for PCR and other diagnostic protocols.
Solid-phase cDNA were first made using oligo-d(T) cellulose powder as a primer matrix for hybridizing specifically with mRNA to recover intact mRNA. Venetainer, P. and Leder, P., Proc. Natl. Acad. Sci. USA, 71:3892-3895, 1974. These cDNA were used only as specialized affinity-matrix for purifying the corresponding complementary mRNA. Oligo-dT ligand was covalently bound, via phospho-enol binding with the 5' phosphate of the oligomer and the hydroxyl group on cellulose sugar residue, to a loose fiber as a cellulose powder. A slurry of this oligo-dT substituted cellulose powder was packed as a column matrix and used for mRNA affinity chromatography. Substrates and reverse transcriptase enzyme were added to the oligo-dT cellulose and annealed to synthesized solid phase cDNA. This mRNA slurry process for making solid-phase cDNA required relatively large quantities of enzyme and mRNA. The process disclosed by Venetainer and Leder is cumbersome and time-consuming.
It is advantageous, therefore, to provide a solid phase matrix which can be used for both mRNA and viral RNA affinity chromatography and as a priming matrix for cDNA synthesis. The resulting solid phase cDNAs will provide a permanent genetic record of the polyadenylated RNAs in a sample, such as from a eukaryotic organism, blood, tumors, donated organs or any other extractable tissue. Repeated extractions of a sample for nucleic acids will be substantially eliminated because the solid-phase cDNAs can be subjected to several different or repetitive genetic analyses without alteration of the nucleic acid sequence. Rare or scarce nucleic acid information may, in this manner, be preserved and archived for subsequent amplification and study. The solid-phase DNAs may be used as sequence templates for genetic analysis, for disease detection and diagnosis, for gene cloning, or for vaccine development.